The DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT) has been shown to protect cells from the toxic effects of alkylating agents including chloroethylnitrosoureas, methylating agents and cyclophosphamide. Most primary tumors have high AGT activity which is primarily responsible for the limited usefulness of these drugs. Severe myelosuppression and therapy-induced myeloid leukemia are life-threatening side effects encountered with the clinical use of alkylating agents. The activity of the AGT protein in human hematopoietic progenitors is very low and it is reasonable to suggest that this is the basis of their high sensitivity to the myelotoxic and mutagenic effects of alkylating agents. In efforts to increase the therapeutic efficacy of alkylnitrosoureas, we have developed O6-benzylguanine (BG). This compound is a potent, selective inactivator of the AGT repair protein that is presently in phase I/II clinical trials as a modulator of alkylating agents. While combining BG with alkylating agents increases killing of a variety of tumor cells, a major concern is that BG will impair the ability of immature hematopoietic cells to repair alkylator-induced damage and that this will, in turn, result in an increase in the incidence of secondary leukemias. In support of this, preliminary data from our phase I trial of BG and BCNU indicates myelosuppression as the dose limiting toxicity. The subject of this proposal is to determine whether BG increases the mutagenicity of alkylating agents which may contribute to the induction of secondary leukemias and more importantly, to use this information to generate therapies to protect against this devastating side effect. Specific aim 1 will address whether inhibition AGT activity results in an increase in mutation frequency at the Hprt locus by cyclophosphamide, BCNU and temozolomide using an in vitro and in vivo model. We will establish whether cells expressing BG-resistant AGT proteins will be protected from BG plus alkylating agent-induced mutations. Specific aim 2 will directly address the question of whether the use of BG with alkylating agents will increase the incidence of therapy related leukemias. Heterozygous Nf1 knockout mice provide an excellent in vivo model of cyclophosphamide-induced myeloid leukemia. Specific aim 3 and 4 describe approaches to combat this devastating side effect. We will generate lines of transgenic mice that over-express mutant AGT to cross with Nf1 mice to determine if there is protection against the incidence of leukemias. In the last specific aim we are proposing the introduction of mutant alkyltransferase genes into hematopoietic stem cells. The introduction of stem cells containing mutant agt genes with high dose alkylating agent chemotherapy will allow us to decrease tumor burden and improve upon the duration of disease free survival and decrease the likelihood of secondary leukemias.